The ultrastructural surface features of tracheal epithelium at various times of development in organ culture were compared with those in the trachea of Syrian golden hamsters of similar age using scanning electron microscopy. Whole tracheal organ cultures, prepared from 3-day-old hamsters, were maintained in HEPES buffered CMRL 1066 medium with 0.2% bovine serum albumin. The ventral epithelial surface of trachea from 3-day-old animals was characterized by numerous microvillous cells, occasional well-developed cilia, and cells containing short cilia representing various stages of ciliary differentiation. After seven days in culture, an increased number of ciliated cells as well as developing cilia were seen. The epithelium after 14 days in culture appeared to have equal numbers of ciliated and microvillous cells, a pattern strikingly similar to that observed in vivo. After 21 days in culture, groups of well-developed cilia were interspersed with nonciliated cells covered with short, poorly developed surface microvilli. A similar pattern was found in the trachea of comparable age (24 days), with the exception of the microvillous cells; many being dome-shaped containing cluster of microvilli with prominent outlines. The tracheal organ culture, as developed in this investigation, appears to represent an excellent model for studying age-related effects of toxic and infectious agents on ciliated epithelial cells.